Restriction endonucleases are capable of recognizing a specific sequence of bases of a deoxyribonucleic acid (DNA) molecule and of enzymatically cleaving in a process termed restriction the double-stranded DNA chain at specific sites. Different restriction endonucleases have an affinity for different recognition sequences. When they are purified away from other contaminating bacterial components, restriction endonucleases can be used in the laboratory to cut DNA molecules into specific fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases, thus, allow the researcher to manipulate the DNA molecule and analyze the resulting construction.
Bacteria usually possess a limited number of restriction endonucleases per species. The endonucleases are named according to the bacteria from which they are derived. For example, the species Proteus vulgaris contains two known restriction endonucleases, named Pvu I and Pvu II. These restriction enzymes recognize and cleave the sequences CGATCG, and CAGCTG respectively. E. coli RY13, on the other hand synthesizes only one enzyme, Eco RI which recognizes the sequence GAATTC.
More than 1000 class-II restriction endonucleases have been identified. See, Kessler et al., Gene, 47:1-153 (1986) and Roberts, Nuc. Acids Res., 17:347-387 (1989) and Kessler et al., Gene, 92:1-248 (1990). The majority of these enzymes recognize four or six basepair palindromic sequences. Thus far, only eight restriction endonucleases with octanucleotide recognition sequences have been found: Asc I (5'-GG/CGCGCC-3', New England Biolabs, Beverly, MA); Fse I (5'-GGCCGG/CC-3', Nelson et al, Nucl. Acids Res., 18:2061-2064 (1990); Not I (5'-GC/GGCCGC-3', Qiang et al., Nucl. Acids Res., 12:4507-4515 (1984); pac I (5'-TTAAT/TAA-3', Polisson et al., unpublished observations; Sfi I (5'-GGCCNNNN/NGGCC-3', where N is either A, C, G or T, Qiang et al., supra; Sgr AI (5'-CR/CCGGYA-3', where R is either A or G and Y is either C or T, Tantz et al., Nucl. Acids Res., 18:3087-3089 (1990); Sse 8287 I (5'-CCTGCA/GG-3', Kotani et al., Nucl Acids Res., 8:5637-5640 (1990); and Swa I (5'-ATTTAAAT-3', Boehringer Mannheim Biochemicals, Indianapolis, IN). Restriction enzymes having these specificities are especially useful for mammalian genome analysis. The purification and characterization of a novel octanucleotide restriction endonuclease, designated Srf I, is the subject of this invention.